Adeno-associated Virus (AAV) is a nonpathogenic, defective human parvovirus. AAV requires that a helper virus co-infect the host cell for efficient virus replication. In the absence of helper virus infection, AAV readily integrates its genome into a specific region in the long arm of chromosome 19. AAV's natural defectiveness, nonpathogenic nature and ability to integrate site-specifically have prompted numerous studies on its potential as a human gene therapy vector. Virus replication and site-specific integration is dependent upon two replication proteins, Rep78 and Rep68, that are encoded by the virus. These proteins mediate virus replication and integration by binding to target sites on the viral DNA and the chromosome 19 locus. These proteins also play important roles in regulation of virus and host cell gene expression. Our laboratory has demonstrated that these proteins are phosphorylated on Ser residues. We propose that differential phosphorylation of Rep78 and Rep68 allow for regulation of these proteins. Preliminary results reveal that the level of phosphorylation varies at different times in the virus replication cycle and that phosphatase inhibition results in an increase in phosphorylation. Experiments proposed here will identify the specific residues that are modified in the Rep proteins and reveal the kinases involved in Rep regulation. We will also determine how specific phosphorylation events modulate the known enzymatic activities of the Rep proteins in in vitro as well as in vivo studies. These investigations will provide essential information required for the understanding of the AAV replication cycle and site-specific integration of AAV gene therapy vectors. These studies may also lead to the development of new and improved AAV vectors.